Animal models of ischemic heart disease

Through the
artificial induction of different forms of cardiovascular pathology, the use of
experimental animals allows for the study of these without interferences
associated with the presence of other diseases or physiological states.

Unmasking the
processes underlying the different manifestations of cardiac failure in a
detailed way requires the use of simplified models. The interaction between
different organs, tissues or cell types is very relevant and contributes to the
maintenance of homeostasis naturally. An event of myocardial ischemia will
often cause a decrease in ejection fraction, and the consequences at the
metabolic level will translate into hormonal and nervous responses that will
again affect the development of coronary heart disease. The development of in vitro models has allowed the study of
specific pathways in isolated cardiomyocytes (Matter, 1969), cardiac fibroblasts
(Leask, 2010) or endothelial cells (Chin, 2011), that are not accessible when
these cells are in their natural environment. However, the need to study
cellular processes in physiologically relevant settings limits the use of these
models. Conversely, the study of ischemic heart disease from human samples is
limited by additional factors that interfere with the findings. Thus, age, sex
or clinical history are difficult to block in studies in which sample size is
limited.

In an animal
model, the induced pathology must faithfully reproduce the structural and
functional characteristics of human pathology. In ischemic heart disease these
involve the chronic narrowing of a coronary artery by deposition of atheroma
plaques or occlusion by thrombosis. There are three possible situations arising
from the decrease in coronary artery diameter.

Chronic ischemia. The occlusion is complete and the flow is not restored.

Myocardial hibernation. The diameter reduction is partial and, in a prolonged or chronic way,
the myocardium has to adapt its metabolism to a deficient supply of oxygen and
nutrients.

Ischemia and reperfusion or transient
ischemia.The vessel occlusion is complete, but the
flow is restored after a period of time.

With this in mind,
one of the models initially considered was to induce the formation of atheromatous
plaques with diets high in fat and cholesterol. This strategy has been
effective in several models (du Toit, 2008; Zhang, 2005). However, the time and
position at which the occlusion occurs is random and for this reason, this
model is suitable for the study of the evolution of atherosclerosis, but is not
practical in experiments aimed to study ischemic heart disease. In addition,
the cardioprotective effect of estrogens has been demonstrated in several
models of hypercholesterolemia (Kolovou, 2011; Clark, 2011), so that gender is
an additional factor to consider that further complicates the development of a
model of ischemic heart disease derived from atherosclerosis.

Although it
obviates other effects associated with atherosclerosis, surgical induction of
occlusion or coronary narrowing greatly facilitates the induction of lesions of
predetermined location and size, which lead to more reproducible results. In
addition, the surgical induction model has the advantage of being able to fix
both the level (total by occlusion or partial by narrowing) and the duration of
ischemia (permanent or followed by reperfusion). Both have advantages and
disadvantages, and each has to be valued depending on the objectives of the
study.

A. TECHNIQUES
FOR SURGICAL INDUCTION OF CARDIAC ISCHEMIA

1. Surgical
induction of chronic myocardial ischemia or hibernation.

(i) Hydraulic occluder and ameroid constrictor.These systems, based
on the total or partial occlusion of a coronary artery branch, are especially used
in large animal models. After an incision in the pericardium, the artery is
exposed and a hydraulic occluder is placed around it, which is inflated to the
desired degree of coronary occlusion. When using an ameroid constrictor, the
casein plastic that composes the device is hydrated at body temperature,
dilating to obtain constriction of the artery. Since the degree of occlusion
can be fixed, both systems are suitable for both chronic ischemia and
myocardial hibernation models.

(ii) Coronary ligation.Following a surgical procedure similar to the
above, the artery is ligated using a thread or umbilical tissue. This system is
used in animal models of very different sizes (Iannini,
1996) but is only suitable for the induction of permanent myocardial ischemia.

(iii) Coronary artery embolism. The strategy of occlusion by coronary embolism
is based on the use of microspheres, agarose, polystyrene beads or
intracoronary injection of thrombin and autologous blood with fibrinogen to
cause coronary obstruction (Sabbah, 1991; Suzuki, 1999). This
procedure is performed in large animals, and the effect obtained is that of permanent
ischemia. Compared to other systems, it has the advantage of being a
percutaneous intervention, which reduces the risk of complications by severe
inflammation. As an obvious limitation is the difficulty of accurately
controlling the exact location of the occlusion.

2.
Surgical induction of ischemia and reperfusion.

(i) Hydraulic occluder and ameroid constrictor.This system has been
described above. The duration of occlusion is a crucial aspect in ischemia-reperfusion
models. Excessive ischemia time may exceed the limit of myocardial numbness and
lead to myocardial infarction.

(ii) Transient coronary ligation.It uses the same
procedure as in the case of permanent ligation. The knot is kept closed during
the agreed time and removed at the end of the operation. In this model, a small
plastic tube is placed between the cord and the vessel, to minimize damage and
allow a better restoration of the coronary flow.

(iii) Transient coronary embolism. The need to use highly
invasive surgical techniques is a disadvantage since the risk of severe
inflammation and other complications makes the survival rate low. In large
animals, transient embolism of the coronary artery can be achieved using
arterial catheterization in a procedure similar to the reperfusion
catheterization performed in patients with acute coronary ischemia events. From
a femoral or radial access, a catheter with an apical balloon is advanced to
the coronary artery, where it is inflated by a hydraulic system to cause total
embolism of the vessel. After the opportune time, the balloon is deflated and
the instruments are removed, suturing the arterial route (van Wijngaarden, 1992).

3. Ex vivo model of ischemia.

In
1895, O. Langendorff developed an ex vivo
cardiac perfusion system. In this model, the heart of the animal selected model
is excised and cannulated, maintaining physiological conditions of temperature and
pH. This model has been used with animals ranging from rat (Bachmann, 1993) to pig (Brenner, 2000) and
allows the study of the effect of drugs, cytokines and other substances without
the interference of endogenous stimuli.

B.
ANIMAL MODELS OF ISCHEMIC HEART DISEASE

Several animal models have been used for the study of ischemic heart
disease. All of them have advantages that make them valuable or practical, and
the selection of the appropriate model must take into account the particular
objectives of each study.

(i)
Models in non-mammalian animals.

Drosophila melanogaster.Despite the phylogenetic distance that
separates it from mammals, the fruit fly presents characteristics that make it
a useful model for the study of molecular mechanisms associated with ischemia. The
signaling pathways associated with hypoxia are very conserved evolutionarily in
D. melanogaster and the catalog of
available mutant strains is exhaustive. Obviously, it is an extremely limited
model when it comes to going beyond gene regulation, but it has been enormously
useful in studies that have revealed, for example, mechanisms of hypoxia
tolerance (Vigne, 2009)
or regulation of cardiomyogenesis (Cripps, 2002).

Danio renio.Its closed cardiovascular system, rapid development, body
transparency and the easiness to establish genetically modified strains make
zebrafish a valuable model in the study of heart disease. D. rhenium presents
the capacity to regenerate, with little or no scar, the cardiac tissue even
when it is extirpated in 20%. After infarction induced by cryogenic damage,
cardiac tissue goes through the stages of mammalian inflammation and fibrosis,
but then the fibrotic tissue matrix is ​​degraded, and cardiomyocytes
proliferate and invade the damaged area to reestablish tissue functionality (Schnabel, 2011; Chablais, 2011). In
contrast to D. melanogaster, D. rhenium is not only susceptible to
surgery, but also extremely tolerant to it.

(ii)
Models in small mammals

Although they have provided very relevant data on the
gene regulation of post-ischemia processes and general cardiac development,
models in non-mammalian animals are limited by the poor clinical relevance of
the results obtained. Much research has focused on small mammal models that
have short breeding cycles, require minor care and, above all, allow the
extensive use of transgenic individuals.

Rodents.
Laboratory rodents like rats and mice have
the advantage of being cheap, homogeneous, easy to breed and genetically
modifiable, while their use is exposed to little ethical debate. These
characterisitic facilitate the use of large sample sizes. The extensive use of
rodents has also led to the development of adapted clinical equipment that
allows an accurate evaluation of parameters such as cardiac function or infarct
sizes. However, there are important physiological differences between these
models and humans (Phoon, 2006). The mouse has a body mass 3000-4000 times lower than
the average adult, a resting heart rate 5 times higherand its metabolic rate is between 10 and 15 times
higher. The action potential of rat and mouse cardiomyocytes is characterized
by being very short and lacking the isoelectric plateau phase (Endoh, 2004). The expression of myosin
isoforms differs between rodents and humans (Haghighi,
2003; Hasenfuss, 1998)and
although the withdrawal of cytosolic Ca2+ occurs due to the activity
of Serca2 in rodents and humans, Na+/Ca2+ exchange is
less relevant in the former (Bers, 2002).

Rabbits.Models in hyperlipidemic
rabbits have been used in several studies of spontaneous myocardial infarction
induced by accumulation of intracoronary plaques, showing that the incidence of
infarction is greater than 90%. In this model, unfortunately, the average
development time is 11 months, and in addition the plaques do not break in a
way equivalent to the human (Shiomi, 2003). As in rodents, other differences at the cellular level
make the rabbit a rather limited model for the study of ischemic heart disease (Hasenfuss, 1998).

(iii) Models in large mammals

Although models in small mammals are appropriate for the study of
processes at the molecular level, several studies show that the conclusions
obtained are not always extrapolated to larger animals (ref267). The heart of larger animals is more similar to human at the anatomical
and physiological levels and therefore, it is sometimes advisable to scale to models
that more accurately reproduce human pathology.

Models in nonhuman primates. Because
non-human primates have greater similarities with man than any other mammal, it
is tempting to think that they would be the best candidates as a model for
cardiovascular disease, but this idea is wrong. Firstly, the use of non-human
primates raises enormous criticism regarding animal rights. This is particularly
relevant on animals with a great genetic similarity with man. It should also be
noted that various primate species are included in the list of endangered species.
In addition, there are remarkable logistical difficulties. Their acquisition
and maintenance are extremely expensive and also harbor potentially infectious
or highly infectious diseases for men, and can be infected by men. Even saving
such considerations, non-human primates have important anatomical and
physiological differences with man. The heart of many primates is very small
and therefore has fewer cardiac cells and a different distribution. These
primates also have very rapid heart rates that can reach 200 beats per minute
at rest (ref268). Finally,
even obviating the aforementioned obstacles, there is no evidence that data
obtained in monkeys provided additional data to those already obtained from
non-primate mammals.

Canine models.Historically, the canine model
has been widely used in the study of cardiac ischemia and myocardial
infarction. Reimer et al. carried out in 1979 a study aimed at accurately
describing the temporal evolution of the lesion in relation to the ischemia
times (ref269). Other
studies have used canine ischemia-reperfusion models to evaluate ventricular
remodeling and its relationship to the renin-angiotensin system (refs270,271) or the effect of stem cell injection on cardiac function (ref272). However, the coronary vascular system of the dog is characterized by
the presence of a significant collateral circulation (refs273,274) which exerts a protective effect on the ischemic damage and alters the
development of the post-ischemic processes, making the size of the infarcts
induced very unpredictable. This has gradually translated research into
alternative models.

Sheep
model. In
models in sheep and pigs, coronary anatomy and the absence of preformed
collateral vessels make it feasible to induce infarctions of predictable size
and location that are adequate for the study of post-ischemic ventricular remodelling (refs274,276). The
ovine model has been used for studies related to the early expansion of the
infarct and the area adjacent to it, showing that border regions extend to adjacent
areas of healthy tissue, involving them in the remodeling process (ref277).
However, as domestic ruminants, sheep have a gastrointestinal anatomy and a
thoracic contour that makes it difficult to obtain ultrasound images and makes
an invasive approach advisable (refs276,277). This
limits in part the potential of this species as models ischemic heart disease.

Porcine
model.Like humans, the pig is an omnivorous animal. Metabolic
rate, heart/body mass ratio, and heart rate are comparable, and blood pressure
is only slightly higher. The pig's heart has a gross anatomy very similar to humans
and has been confirmed in the work of several authors as a model suitable for
the study of the pathophysiology of ischemic heart disease (refs279,280), but also studies focused on dilated cardiomyopathy (ref281)or cell theraphy (ref272) support the validity of this model in the
cardiovascular field. Because of the similarities found between the hearts of
both species, the pig has been used even as a donor in some of the rare
experiences of cardiac xenotransplantation (ref282) and cardiac valves are currently used as
xenografts in clinical practice (ref283). The main limitations of the use of this
animal model have to do with a certain predisposition to the development of
arrhythmogenesis in models of ischemic heart disease, which is nevertheless
overcome by the administration of supplements with electrolytes and
anti-arrhythmogenic agents in non-invasive models (ref284). The limitation represented until recently
by the lack of databases and complete catalogs for the pig at the genomic and
proteomic level appears increasingly reduced with the sequencing of the porcine
genome (ref285,286) and the increase in the number of available
antibodies that recognize porcine molecules. For a more detailed discussion on
the differences and similarities between the human and the porcine heart,
please visit this link.

C.
ETHICAL CONSIDERATIONS REGARDING THE USE OF ANIMAL MODELS

Every year, more than 75
million vertebrates are used worldwide for experimental purposes (ref292), being
mice and rats the most widely used species. Experiments using animals must be
designed under the "Principle of the Three R". 1) Replacementof live animals by in vitro
or computerized models. 2) Reduction in the
number of animals used for experiments. To this end it is necessary to use of
standardized animals that reduce biological variability, and to make previous
estimates of statistical power. 3) Refinement, which means guaranteeing maximum animal comfort, by
providing sufficient care that covers physiological and ethological animal needs,
and avoiding unnecessary suffering through the use of adequate anaesthesia (ref293). In summary, the use of
experimental animals should be considered only when there is no other viable
alternative. It is necessary to take into account that the discomfort and
stress during the experiments, as well as an unreasonable experimental design,
are a violation of animal rights, but also inevitably lead to non-specific
effects that will ultimately distort the results (ref293).